This proposal responds to PA-02-073, "Innovation Grants for Research in Human Immunology." The purpose of these studies is to better understand the relationship between CD8+ T-cell responses, viral replication and CD4+ T-cell depletion in gut-associated lymphoid tissue (GALT), and to clarify the ability of GALT T-cells to perform antiviral immune effector functions. We have developed methods to assess the phenotype and function of antigen-specific T-cells freshly isolated from human rectal tissue. For SPECIFIC AIM 1, we will assess the frequency, breadth and memory/effector phenotype of HIV-specific CD8+ T-cells in GALT and blood. Blood and rectal biopsy samples will be obtained from (i) a cross-sectional study of 30 HIV-infected men and women (including 15 "on" and 15 "off" HAART) and 10 controls; (ii) a longitudinal study of 20 HIV-infected individuals (10 "on" HAART, 10 "off'), with samples taken at 6-month intervals. We hypothesize that in patients not on HAART the frequency and breadth of CDS+ T-cell responses in GALT may exceed that in blood. In patients on HAART, we predict a decline in the magnitude and breadth of these responses concurrent with decreased tissue viral load. We also predict that reduced viral load and CD4+ T-cell repopulation will provide partial reconstitution of CD4+ T-cell responses to non-HIV antigens in GALT. For SPECIFIC AIM 2, we will assess the effector functions of HIV-specific CDS+ T-cells in GALT. Preliminary results suggest that (i) HIV-specific CTL in blood express low levels of perforin, (ii) perforin expression by all CD8+ T-cells is decreased in GALT relative to blood, and (iii) granzyme A and perforin are differentially expressed. We hypothesize that CD8+ T-cells in GALT may be functionally impaired. We will assess effector functions of HIV- and CMV- specific CTL in GALT by examining rectal biopsy specimens obtained in a cross-sectional study of 30 HIV-infected men and women (15 "on" and 15 "off' HAART) and 10 controls. GALT CD8+ T-cells will be assessed for production of perforin, granzymes A and B, MIP-1beta, IL-2, TNF-alpha and IFN-gamma, and for their ability to kill target cells in an MHC class I-restricted manner. We will also test alternate hypotheses to explain the apparent low levels of perforin expression in GALT.